U.S. patent number 6,911,565 [Application Number 10/255,362] was granted by the patent office on 2005-06-28 for process for the production of xylitol.
This patent grant is currently assigned to Danico Sweetners Oy. Invention is credited to Heikki Heikkila, Andrei Miasnikov, Heikki Ojamo, Vili Ravanko, Matti Tylli.
United States Patent |
6,911,565 |
Heikkila , et al. |
June 28, 2005 |
**Please see images for:
( Certificate of Correction ) ** |
Process for the production of xylitol
Abstract
The present invention relates to a process for the production of
xylitol. The process utilises ribulose for the preparation of
xylitol and involves several different conversion reactions, such
as reduction, epimerisation and/or isomerisation. The present
invention also relates to the use of ribulose for the preparation
of xylitol.
Inventors: |
Heikkila; Heikki (Espoo,
FI), Ojamo; Heikki (Kirkkonummi, FI),
Miasnikov; Andrei (Kantvik, FI), Ravanko; Vili
(Clinton, IA), Tylli; Matti (Kantvik, FI) |
Assignee: |
Danico Sweetners Oy (Espoo,
FI)
|
Family
ID: |
8561956 |
Appl.
No.: |
10/255,362 |
Filed: |
September 26, 2002 |
Foreign Application Priority Data
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Sep 26, 2001 [FI] |
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20011889 |
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Current U.S.
Class: |
568/852; 435/158;
435/190; 435/94; 536/124; 536/127; 536/128; 568/861; 568/864 |
Current CPC
Class: |
C12P
7/18 (20130101); C07C 29/132 (20130101); C07C
29/132 (20130101); C07C 31/18 (20130101); C07B
2200/07 (20130101) |
Current International
Class: |
C07C
29/00 (20060101); C07C 29/141 (20060101); C07C
29/145 (20060101); C12P 7/02 (20060101); C12P
7/18 (20060101); C07C 031/18 (); C07C 031/20 ();
C07C 031/22 (); C07C 031/24 (); C07C 031/26 () |
Field of
Search: |
;568/852,861,864
;435/94,158,190 ;536/124,127,128 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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0 716 067 |
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Jun 1996 |
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EP |
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0807682 |
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Nov 1997 |
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EP |
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0 745 758 |
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Feb 1998 |
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EP |
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1 518 510 |
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Mar 1968 |
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FR |
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2 641 545 |
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Jul 1990 |
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FR |
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2 772 788 |
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Jun 1999 |
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FR |
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WO 91/15588 |
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Oct 1991 |
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WO |
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WO 93/01299 |
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Jan 1993 |
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WO |
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WO 93/19030 |
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Sep 1993 |
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WO |
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WO 88/05467 |
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Jul 1998 |
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WO |
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WO 01/53306 |
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Jul 2001 |
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WO |
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Other References
Gong, Cheng-Shung, et al. "Quantative Production of Xylitol from
D-xylose by a High-Xylitol Producing Yeast Mutant", Biotechnology
Letters,3(3): 130-135 (1981). .
Kuhad, Ramesh C., et al. "Optimization of xylanase production by a
hyperxylanolytic mutant strain of Fusarium oxysporum", Process
Biochemistry, 33(6): 641-647 (1998). .
Itoh, Hiromichi, et al., "Purification and Characterization of
D-Tagatose 3-Epimerase from Pseudomanas sp. ST-24", Biosci.
Biotech. Biochem., 58(12): 2168-2171 (1994). .
Bruijn, J.M., et al., "Reactions of Monosaccharides in Aqueous
Alkaline Solutions", Sugar Technology Reviews, 13: 21-52 (1986).
.
Bhuiyan, Shakhawat, et al. "A New Method for the Production of
L-Lykose from Ribitol Using Microbial and Enzymatic Reactions",
Journal of Fermentation and Bioengineering, 86(5): 513-516 (1998).
.
Matsui, Masanao, et al., "Studies on 2-Keto-D-gluconic Acid", Agr.
Biol. Chem., 27(3): 180-184 (1963). .
Abstract of Japanese Laid-Open Application No. 11113567, published
Apr. 27, 1999. .
Abstract of Japanese Laid-Open Application No. 8056659, published
Mar. 5, 1996..
|
Primary Examiner: Price; Elvis O.
Attorney, Agent or Firm: Scully, Scott, Murphy &
Presser
Claims
What is claimed is:
1. Process for the preparation of xylitol from ribulose comprising
the steps of: epimerizing and isomerizing ribulose to form a
mixture of ribulose, xylulose and xylose; chromatographically
separating a xylose-rich fraction; and reducing said xylose-rich
fraction into a xylitol-rich fraction.
2. Process for the preparation of xylitol from ribulose comprising
the steps of: reducing and epimerizing ribulose to a mixture of
ribitol, arabitol and xylitol; and chromatographically separating
of a xylitol-rich fraction.
3. Process according to claim 2, comprising catalytic epimerisation
of alditols.
4. Process according to claim 1 or 2, wherein a mixture of ribulose
and xylulose is used as starting material.
5. Process according to claim 1 or 2, wherein part of the fractions
obtained in said chromatographic separation step are recirculated
into the isomerisation and/or epimerisation.
6. Process according to claim 5, wherein the fractions containing
ribulose and xylulose are recirculated.
7. Process according to claim 1 or 2, wherein said epimerization
and isomerization steps are carried out simultaneously or in
series.
8. Process according to claim 7, wherein epimerisation is carried
first, followed by isomerisation.
9. Process according to claim 7, wherein isomerisation is carried
out first, followed by epimerisation.
10. Process according to claim 1 or 2, wherein ribulose is prepared
by fermentation.
11. Process according to claim 1 or 2, wherein xylose is reduced to
xylitol by hydrogenation.
12. Process according to claim 1 or 2, wherein xylitol is
recovered.
13. Process according to claim 1 or 2, wherein xylitol is recovered
by crystallization.
14. Process according to claim 3 wherein said epimerization and
isomerization steps are carried out simultaneously or in
series.
15. Process according to claim 4 wherein said epimerization and
isomerization steps are carried out simultaneously or in
series.
16. Process according to claim 10 wherein glucose is fermented to
produce said ribulose.
17. Process according to claim 3 wherein xylose is reduced to
xylitol by hydrogenation.
18. Process according to claim 4 wherein xylose is reduced to
xylitol by hydrogenation.
19. Process according to claim 3 wherein xylitol is recovered by
crystallization.
20. Process according to claim 4 wherein xylitol is recovered by
crystallization.
21. Process according to claim 1 comprising recovering xylose from
said xylose-rich fraction by crystallization; and converting said
crystalline xylose to xylitol.
Description
FIELD OF THE INVENTION
The present invention relates to a process for the production of
xylitol. In particular, the invention relates to a process for the
production of xylitol comprising conversion of ribulose.
BACKGROUND OF THE INVENTION
Xylitol is a naturally occurring 5-carbon sugar alcohol, which is
present in small amount in many fruits and vegetables and is
produced in the human body during normal metabolism. It has
approximately the same sweetness as sucrose, lower caloric content
of about 2.4 kcal/g, and it has certain known metabolic, dental and
technical characteristics which make it an attractive special
sweetener or sugar substitute in various contexts. For instance,
xylitol is cariostatic and even anti-cariogenic. It is metabolised
independently of insulin and can be safely consumed by non-insulin
dependent diabetics, and it is non-toxic. Nowadays it is widely
used in chewing gums, dental care products, health promoting
products, functional food products, pharmaceutical products,
confectionery products and the like.
Xylitol is usually prepared by processes utilizing natural raw
materials, especially xylan-containing materials. In current use
are methods in which a xylan-containing material is first
hydrolysed to produce a mixture of monosaccharides, including
D-xylose. After purification the D-xylose is then converted to
xylitol, generally in a chemical process using e.g. a nickel
catalyst such as Raney-nickel. A number of processes of this type
have been described in the literature of the art. U.S. Pat. No.
3,784,408 (Jaffe et al.), U.S. Pat. No. 4,066,711 (Melaja et al.),
U.S. Pat. No. 4,075,406 (Melaja et al.), U.S. Pat. No. 4,008,285
(Melaja et al.) and U.S. Pat. No. 3,586,537 (Steiner et al.) may be
mentioned as examples.
The recovery of D-xylose during wood and pulp processing can be
performed by various separation techniques. Chromatography is
widely used. A process for the fractionation of sulfite spent
liquor by chromatography is described in U.S. Pat. No. 5,737,225,
Xyrofin Oy. The process uses a simulated moving bed including at
least two chromatographic beds and, preferably, at least three
different fractions are recovered, one of these being enriched with
xylose and another with lignosulphonates. For instance U.S. Pat.
Nos. 4,631,129; 4,008,285 and 4,075,406 also describe
chromatographic processes for the recovery of xylose.
Processes in which microorganisms are utilised for biotechnological
production of xylitol have also been described. It is known that
many yeast strains produce reductase enzymes that catalyse the
reduction of sugars to corresponding sugar alcohols. Many yeasts,
in particular Pichia, Candida, Hansenula and Kluyveromyces, are
also capable of reducing xylose to xylitol as an initial step in
their xylose metabolism.
The reaction route or pathway of xylose utilisation for yeasts is
in general the following: xylitol is synthesised in the first step
by reduction of xylose to xylitol with the aid of xylose reductase.
Xylitol is then metabolised by a series of successive steps.
Xylitol is first oxidised to xylulose with xylitol dehydrogenase,
xylulose is phosphorylated to xylulose-5-phosphate with xylulose
kinase (also called xylulokinase), and then part of the
xylulose-5-phosphate is converted to pyruvate via several
intermediate steps. Also ethanol and CO.sub.2 can be formed. The
reactions are not tightly coupled, and the relevant main products
and by-products vary depending on the yeast strain and the
fermentation conditions, such as oxygen availability.
For instance PCT publications WO 90/8193, WO 91/0740, WO 88/5467
and French published application 2 641 545 describe the use of
Candida tropicalis, Candida guilliermondii and Candida
parapsilosis, respectively, for the industrial production of
xylitol.
U.S. Pat. No. 5,081,026, Heikkila et al., describes a process for
the production of xylitol from xylose, in which an aqueous xylose
solution is fermented with a yeast strain capable of converting
free xylose to xylitol and free hexoses to ethanol. After
fermentation, a xylitol-rich fraction is obtained by
chromatographic separation, and finally, xylitol is recovered from
said fraction.
Genetic modification of microorganisms in order to enhance their
xylitol production have also been reported in the literature of the
art. For example, in WO 91/15588, Hallborn, J. et al. describe the
cloning of the xylose reductase gene from Pichia stipitis into
Saccharomyces cerevisiae. Gong C. et al., Biotechnol. Letters
3:125-130 (1981) describe two high xylitol producing yeast mutants
denominated HXP1 and HXP2, obtained after UV-mutagenesis of a wild
strain of Candida tropicalis which originally was capable of
metabolising D-xylose into xylitol.
EP 0 604 429, Xyrofin, describes novel yeast strains with modified
xylitol metabolism, a process for the production of said strains,
and the use of said strains in a process for producing xylitol. The
strains are capable of reducing xylose into xylitol, but are
deficient in one or more enzymes involved in the xylitol
metabolism, with the effect that the xylitol produced accumulates
in the culture medium and can be recovered therefrom. The yeasts
described belong to the genera Candida, Hansenula, Kluyveromyces or
Pichia, and the genetic modification eliminates or reduces
expression of the gene that encodes xylitol dehydrogenase or
xylulose kinase, or both.
Another approach that has been suggested for the bioproduction of
xylitol is the enhancement of xylose production, thus providing
more xylose as the primary metabolite for xylitol production.
Some fungi, including Aureobasidium, Aspergillus, Trichoderma,
Fusarium and Penicillium, have been reported to have xylanolytic
activity and thus be able to degrade xylan-containing biopolymers
into xylose. E.g. Kuhad R. C. et al., Process Biochemistry
33:641-647 (1998) describe a hyperxylanolytic mutant strain of
Fusarium oxysporum produced by UV and
N-methyl-N'-nitro-N-nitrosoguanidine (NTG) treatment.
EP 0 672 161, Xyrofin, describes a method for the production of
xylitol from carbon sources other than xylose and xylulose by using
recombinant hosts. The microorganisms produce xylitol via an
altered arabitol route involving in particular arabitol
dehydrogenase, and/or via altered (over)expression of genes
encoding the enzymes of the oxidative branch of the pentose
phosphate pathway (PPP), in particular glucose-6-phosphate
dehydrogenase or 6-phospho-D-gluconate dehydrogenase, thus enabling
utilisation of glucose, for instance. When used, D-glucose is
phosphorylated into D-glucose-6-phosphate and converted to
D-ribulose-5-phosphate via 6-phospho-D-gluconate. The
D-ribulose-5-phosphate is then epimerised to
D-xylulose-5-phosphate, dephosphorylated to D-xylulose and reduced
to xylitol. Amplification of glucose-6-phosphate dehydrogenase
enzyme activity in osmotolerant yeasts is later also described in
FR 2 772 788, Roquette Freres.
U.S. Pat. No. 5,096,820, Leleu et al., also describes a process in
which xylitol is produced from D-glucose. Glucose is first
microbiologically converted to D-arabitol, which likewise is
microbiologically converted to D-xylulose. The D-xylulose is then
enzymatically isomerised into a mixture of D-xylose and D-xylulose,
which is catalytically hydrogenated. Finally, the xylitol is
recovered by chromatographic separation or crystallisation. The
D-arabitol containing fractions, or the mother liquor from
crystallization, which are rich in xylitol but also in D-arabitol,
are preferably recirculated into the process. U.S. Pat. No.
5,238,826, Leleu et al., uses a similar process to obtain D-xylose,
ultimately for the preparation of xylitol by hydrogenation. Also in
this process, D-glucose is first microbiologically converted to
D-arabitol, which then likewise is microbiologically converted to
D-xylulose. The D-xylulose is then enzymatically isomerised into a
mixture of D-xylose and D-xylulose. Finally, the mixture is
subjected to chromatographic separation, the D-xylose fraction is
recovered and the D-xylulose fraction is recirculated into the
isomerisation step.
The background art thus describes the production of xylitol from
naturally occurring raw materials. At present, the raw materials
mainly used are xylan-containing materials. From xylan, xylitol is
produced by chemical processes or combinations of chemical and
biological processes. Further, processes utilising microorganisms,
in particular yeasts, capable of producing xylitol from
monosaccharide solutions or pure D-xylose solutions have been
described.
In view of the increasing use of xylitol, in particular due to its
properties as sweetener and therapeutic effects, new methods for
the production of xylitol would be welcome. In particular, there is
an expressed need for processes for the production of xylitol from
other sources than those mainly utilised.
U.S. Pat. No. 5,714,602, Cerestar Holding B.V., discloses a process
developed from the this viewpoint. According to the document,
xylitol is produced from gluconic acid. In a first step, gluconic
acid is decarboxylated, by using sodiumhypochlorite or hydrogen
peroxide into arabinose, which through hydrogenation is converted
into arabinitol. After epimerisation, a mixture of xylitol, ribitol
and D,L-arabinitol is obtained, from which xylitol is recovered by
chromatographic methods.
EP 754 758, Cerestar Holding B.V., relates to a process for the
production of xylitol in two steps. In the first step a hexose is
converted to a pentitol by fermentation, and in the second step the
pentitol is catalytically isomerised to yield a pentitol mixture.
Specifically, the document describes a process in which glucose is
fermented to arabinitol and then isomerised into a pentitol mixture
containing xylitol, ribitol and D,L-arabinitol. Xylitol can be
recovered from said mixture by chromatographic methods.
WO 93/1903, Amylum, also describes a process for the production of
xylitol from monosaccharides, in particular D-glucose, D-fructose,
D-galactose, L-sorbose or mixtures thereof. The starting material
is first oxidized to D-arabinonic acid, D-lyxonic acid, and/or
L-xylonic acid and the intermediate is then hydrogenated in one or
several steps to a product consisting mainly of xylitol or a
mixture of xylitol, arabinitol and ribitol. Finally, if necessary,
xylitol is separated by means of chromatography.
SHORT DESCRIPTION OF THE INVENTION
The present invention is based on the utilisation of ribulose for
xylitol production. Surprisingly, it has been found that xylose can
be produced from ribulose by a simple process comprising at least
one of the following two conversion reactions, epimerisation and
isomerisation. Eventually, xylose is converted to xylitol.
It is thus an object of the present invention to provide a process
for the preparation of xylitol from ribulose by reduction and at
least one of epimerisation and isomerisation.
In a preferred embodiment of the invention, the process for the
preparation of xylitol utilises a mixture of ribulose and xylulose
as starting material.
In another preferred embodiment, the process also involves at least
one separation step. In particular, chromatographic separation is
used.
In still a preferred embodiment of the present invention, some of
the fractions, in particular xylulose- and ribulose-containing
fractions, obtained in the separation step(s) are recirculated into
the isomerisation and/or epimerisation steps.
In accordance with the present invention, the processes can be
carried out chemically, microbiologically, or enzymatically.
Further, the reactions can be carried out simultaneously, in
parallel or sequentially.
The present invention also describes processes for the preparation
of ribulose. Also processes for the preparation of mixtures of
ribulose and xylulose are described.
Furthermore, processes for the purification and recovery of the
products are described. Preferably, xylose and xylitol are
recovered by crystallization.
Still further, the invention relates to the use of ribulose for the
preparation of xylitol.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is thus based on utilisation of ribulose.
Previously, ribulose has been regarded as disadvantageous in
xylitol production. Fermentation is widely used as a first step in
the process, and fermentation always yields a mixture which is
problematic when considering the further processing. This is
particularly the case when using glucose as the starting material
for fermentation. In accordance with the present invention, instead
of being a harmful by-product in the process, ribulose can be
utilised as an advantageous starting material for xylitol
production. In particular, xylitol can be produced from both
xylulose and ribulose, without experiencing problems in the
xylulose-xylose route.
The process according to the invention utilises at least two steps,
reduction, epimerisation and/or isomerisation for conversion of
ribulose. In the process, both D- and L-ribulose can be used.
In connection with the present invention, isomerisation is defined
as an equilibrium reaction between aldoses and ketoses, and
epimerisation is defined as an equilibrium reaction between
ketoses.
The epimerisation reaction can preferably be carried out by using
an enzyme having ketose 3-epimerase (tagatose epimerase) activity,
and thus being able to convert ribulose to xylulose. A suitable
enzyme for carrying out the epimerisation process has been
described for instance in U.S. Pat. No. 5,411,880, Izumori et al.,
disclosing the enzyme D-keto-hexose 3-epimerase obtained from
Pseudomonas cichorii ST-24, FERM BP-2736. The enzyme epimerises
D-ketohexoses, D-ketopentoses and L-ketopentoses at their C-3
positions to form their corresponding epimeric counterparts. The
examples show e.g. the interconversion reactions of D-xylulose and
D-ribulose, and L-xylulose and L-ribulose, respectively. In Biosci.
Biotech. Biochem. 58 (1994) 2168-2171, where the inventors are
coauthors, the same enzyme is described but renamed as D-tagatose
3-epimerase. It is pointed out that the enzyme epimerises free
keto-sugars but not phosphorylated sugars, and is more active on
ketoses with cis than trans configuration.
The reaction can be carried out by using both isolated and purified
enzyme preparations as well as microorganisms expressing such
enzyme activity. The gene for tagatose epimerase is known and has
been successfully cloned and expressed e.g. in E. coli (Ishida, Y.,
Kamiya, T., Itoh, H., Kimura, Y., Izumori, K. J. Ferment. Bioeng.
83 (1997) 529-534). Suitable production systems can hence also be
constructed by genetic engineering.
The ketose 3-epimerase enzyme is able to epimerise several
hexuloses and pentuloses at their 3-carbon. Due to the
reversibility of the reaction, a mixture is usually formed.
Starting with D-ribulose, a mixture of D-ribulose and D-xylulose is
formed, which in equilibrium contains about 85% D-xylulose. The
same equilibrium is also formed when starting from a mixture of
D-ribulose and D-xylulose.
The isomerisation reaction can be carried out chemically,
microbiologically, or enzymatically. For enzymatic isomerisation,
an enzyme having glucose isomerase or xylose isomerase acticity can
be used. The enzyme catalyses the conversion of xylulose to xylose.
Such enzymes are commercially available. As one example, Spezyme
GI, Genencor Int., Finland, may be mentioned. It has been analysed
that Spezyme Gl effects some amounts of conversation between
ribulose and ribose. Preferably, an enzyme that does not isomerise
D-ribulose is used. Also this reaction can naturally be carried out
by using both isolated and purified enzyme preparations and natural
or genetically engineered microorganisms expressing such enzyme
activity. Chemically, the reaction can be carried out for instance
by alkaline isomerisation, as described e.g. by de Bruijn et al.,
SugarTechnology Reviews 13 (1986) 21-52.
For enzymatic conversions, immobilized or free enzymes can be used,
for instance in continuously stirred tank reactions (CSTR).
Preferably, the conversions are carried out with immobilised
enzymes in continuous flow columns. The reactions are reversible
and the compounds and yields obtained will depend on the
equilibrium constants and the conditions used. Usually, compound
mixtures are obtained. Both enzymatic steps, however, favour the
formation of the preferable products. The epimerisation and
isomerisation reactions can be performed sequentially in either
order, in parallel or simultaneously.
The reduction reaction can also be performed chemically,
enzymatically or microbiologically. Chemical reduction, by using
e.g. hydrogenation, metal borohydride, amalgam, or electrochemical
reduction is regarded as preferred. Most preferably, catalytic
hydrogenation is used.
Preferably, the process according to the invention also comprises
at least one separation step. Preferably, chromatographic
separation is used. The chromatographic separation can be performed
at different stages of the process, and the process can involve
several different separation steps. When, for instance, the process
according to the invention is carried out by using
ribulose/xylulose as a starting material, and performing
isomerisation as the first step, a mixture of ribulose, xylulose
and xylose is obtained. From this mixture, xylose can be separated,
whereafter an epimerisation reaction may be carried out on the
remaining mixture of ribulose and xylulose. In addition to
chromatographic separation, fractions can be separated or purified
by using crystallization, ion exchange, membrane processes, such as
ultrafiltration, nanofiltration, or electrodialysis, for
instance.
Alternatively, the order can be reversed, but the separation step
conserved. In such a case, epimerisation of ribulose/xylulose will
give a mixture enriched with xylulose. After separation of
ribulose, isomerisation is carried out on the xylulose
fraction.
A still further alternative is to carry out epimerisation and
isomerisation first, and optionally carry out separation as a final
step.
The reactions can thus be carried out sequentially in the desired
order. It is also possible to perform the reactions simultaneously,
for example in a CSTR or continuous flow column containing the
enzymes or microbes needed.
The process according to the present invention can also involve
other reactions, for example catalytical alditol epimerisation.
A preferred alternative is to recirculate products obtained in the
reactions back into the process.
For instance, ribulose, or a mixture of ribulose and xylulose, can
be epimerised, whereby a mixture enriched with xylulose, but also
containing ribulose, is obtained. As shown in the examples, the
mixture can contain different amounts of ribulose and xylulose, for
instance about 15% of ribulose and about 85% of xylulose.
Isomerisation of said mixture will yield a mixture of xylose,
xylulose and ribulose. Starting from the relative proportions
mentioned above, e.g. a mixture of 70% xylose, 15% of xylulose and
15% of ribulose can be obtained. Said components can be separated,
for instance by chromatographic separation, yielding a fraction
enriched with xylose, a fraction containing ribulose and a fraction
containing xylulose. The xylose fraction can be purified and xylose
recovered therefrom e.g. by crystallization. The xylose can also,
directly or in purified form, be hydrogenated into xylitol. The
ribulose fraction is preferably recirculated back to the
epimerisation reaction, and the xylulose fraction is preferably
recirculated back to the isomerisation reaction.
Each of the mixtures can be hydrogenated, the hydrogenation
resulting in a mixture of ribitol, arabitol (=arabinitol) and
xylitol, from which xylitol can be separated and recovered.
Fractions containing ribitol, arabitol and/or xylitol, obtained in
said separation, can be recirculated back to the hydrogenation
step. The starting material, ribulose, can also be isomerised to
ribose, and then hydrogenated and epimerised to ribitol, xylitol,
DL-arabitol containing pentitol mixture as already described.
A further alternative is to subject the ribulose/xylulose mixture
obtained after epimerisation, or fermentation, to a separation
process. The ribulose containing fraction obtained in said
separation is then circulated back into the epimerisation process,
and the xylulose containing fraction obtained is circulated back
into the isomerisation process for production of xylose, or used
for hydrogenation. Hydrogenation of xylulose results in a mixture
of arabitol and xylitol. These components can be separated from
each other e.g. by chromatographic separation as described in the
examples and as known from the literature for example (U.S. Pat.
No. 4,008,285).
The epimerisation and isomerisation of ribulose yield xylose as the
main product. Other products, such as xylulose, ribitol, and
arabitol, obtained during the process can also be recovered.
However, these products can also be recirculated into the process
in order to improve the xylose/xylitol yield. The xylose obtained
is eventually converted to xylitol by enzymatic or microbiological
reduction. Chemical reduction, by using e.g. hydrogenation, metal
boro hydride, amalgam, or electrochemical reduction, is regarded as
preferred. Most preferably, catalytic hydrogenation is used. Before
the chemical reduction reactions the solutions can be purified e.g.
by using ion exchange methods in order to improve the reaction
performance, i.a. for extending the life time of the catalyst.
The starting material for the process is ribulose. A mixture of
ribulose and xylulose can also be used.
Some microorganisms are able to produce ribulose. Ribulose is thus
a naturally occurring compound. However, processes for preparing
ribulose in industrial scale have been scarcely reported, and
ribulose is not commercially available.
Ribulose, and xylulose, can be prepared from D-glucose for instance
by microbiological fermentation. D-glucose is an abundant natural
compound and commercially available at low cost. D-glucose is thus
a preferred raw material for ribulose preparation.
In connection with the present invention, some Bacillus strains
have been shown to convert glucose with 30-50 wt % yields to a
mixture of D-ribulose and D-xylulose. Such strains can be obtained
by screening natural mutants, by conventional mutagenesis, and by
genetic engineering. Strains that have been genetically engineered
for the purposes of the present invention have been shown to
produce ribulose/xylulose 2:1 mixtures in a yield of about 35 wt %
in non-optimised conditions.
Ribulose, and xylulose, can also be obtained for instance by
isomerisation of ribose. Isomerisation can be performed both
chemically, microbiogically, and enzymatically. A suitable method
for isomerisation of L-ribose has been described e.g. in U.S. Pat.
No. 6,037,153, Hayashibara Biochem Lab. Production of D-ribose has
been described in U.S. Pat. No. 3,607,648, Takeda Chem. Ind.
Ltd.
Other sources for ribulose and xylulose production are for instance
2-keto-L-gulonic acid and 2-keto-D-gluconic acid.
In connection with the present invention, the starting material is
preferably produced by fermentation.
Examples of suitable agents and methods for carrying out the
appropriate reactions are disclosed in the literature of the art.
For instance, production of D-ribulose and D-xylulose by
fermentation with Brevibacterium sp., ATCC 21049, and
Corynebacterium sp., ATCC 21050, is described in FR 1,518,510,
Kyowa Ferm. Ind. Co., Ltd and CA 840,981, Kyowa Hakko Kogyo Co. The
strains are commercially available. As examples of suitable carbon
sources, saccharides, such as glucose, fructose, maltose, sucrose,
starch, starch hydrolysate, molasses, and glycerol, mannitol,
sorbitol, and organic acids are mentioned.
A ribitol dehydrogenase capable of forming D-ribulose from ribitol
in the presence of NAD+ has been described in JP 8056659,
Hayashibara Seibutsu Kagaku. A thermostable D-arabinitol
dehydrogenase capable of oxidizing D-arabinitol to D-ribulose has
been described in JP 11113567, Ikeda Shokuken KK and Nippon Kayaku
Ltd. The enzyme is, however, suggested for use as a diagnostic for
candidiasis. A dehydrogenase gene from Pichia stipitis having
D-arabinitol dehydrogenase activity and capable of producing
D-ribulose from D-arabinitol has been described by Hallborn et al.,
Yeast (England) July 1995, 11 (9) pp. 839-847.
JP 11018792, Mitsubishi Chem Corp, describes micrororganisms
capable of converting ribitol to L-ribulose. The microorganisms
mentioned belong to the genus Gluconobacter, Acetobacter,
Alcaligenes and Acinetobacter; Gluconobacter frateurii IFO 02508 is
mentioned as preferred. Mutants of Klebsiella aerogenes W70 which
are constitutive for L-fucose isomerase have been shown to produce
D-ribulose from D-arabinose by Charnetsky et al. J. Bacterol. 119
(1974)162-169. A thiamine-requiring Corynebacterium strain
producing D-ribulose from gluconic acid has been described in JP
45039034, Godo Shusei Co., Ltd.
Stereospecific oxidation of polyols and sugars to form
corresponding ketoses and carbonyl sugars, respectively, has been
described by Huwig et al., Meded. Fac. Landbouwwet. Rijksuniv.
Gent. 59 (1994)2393-2401. The bioconversion reactions were
performed by using Pseudomonas sp. L-glucitol dehydrogenase (GDH)
and Peniophora gigantea immobilised pyranose oxidase (PO, EC
1.1.3.10). By using L-arabitol as the substrate, 60% conversion to
L-xylulose/L-ribulose (4:1) was obtained.
Shakhawat et al., Journal of Fermentation and Bioengineering 86
(1998) 5, pp. 513-516, describe the preparation of L-lyxose from
ribitol by a microbial oxidation reaction, which yields L-xylulose
as an intermediate.
PCT EP99/09771, Xyrofin Ltd., describes a process for the
production of L-ribose from L-arabinose. The L-ribose can be used
for the preparation of L-ribulose e.g. by isomerisation.
Decarboxylation of 2-ketoaldonic acids has been described by
Matsui, M, Uchiyama, M. and Liau, Agr. Biol. Chem. 27(1963) 3, p.
180-184. The authors present a scheme for the decarboxylation of 2-
and 3-ketoacids into corresponding ketoses and aldoses, and
elaborate on the importance of a metal ion catalyst. Nickel ion
catalysed decarboxylation of 2-keto-D-gluconic acid in pyridine
yielded D-ribulose as the main product. In addition, D-arabinose
was found in a smaller amount. Decarboxylation of 2-keto-L-gulonic
acid, on the other hand, yielded L-xylose and L-xylulose in
approximately equal amounts. It is mentioned that D-arabinose and
L-xylose possibly are formed from the main products D-ribulose and
L-xylulose due to an alkaline isomerisation process mediated by the
pyridine used. An improved method avoiding both the use of pyridine
which is harmful in itself, and the isomerisation process, is
described in U.S. Pat. No. 5,872,247, Duflot, P. and Fleche, G. The
nickel ion catalysed decarboxylation process is performed by
putting an aqueous solution of 2-ketoaldonic acid in contact with a
resin carrying vinylpyridine groups. According to the document, the
process allows for obtaining the ketose of the corresponding
functionality immediately lower than the ketoaldonic acid in high
yield and purity. D-ribulose is disclosed as the decarboxylation
product of 2-keto-D-gluconic acid, D-xylulose of
2-keto-D-galactonic acid, and D-erythrulose of 2-keto-D-arabinonic
acid, respectively.
The ribulose prepared can be separated and purified. It can be
used, for instance, for the preparation of ribitol.
In connection with the present invention, the ribulose obtained is
preferably utilised for the production of xylose and xylitol as
outlined above.
The invention will be described in detail in the following specific
examples. The examples are included herein for illustrative
purposes only and are not to be construed as limiting or
restricting the scope of the invention in any way.
EXAMPLE 1
Production of a D-xylulose/D-ribulose Mixture
The mutant Bacillus subtilis strain GX7 described in PCT patent
application PCT/FI01/00051 was cultivated on a LB (Luria broth)
medium containing 50 g/l glucose under aerobic conditions at
30.degree. C. in a shake flask. After 120 hours the glucose was
converted to 9.8 g/l D-ribulose and 3.3 g/l D-xylulose. The
cellmass was then separated from the fermentation broth by
centrifugation and the clarified broth was concentrated under
vacuum to 1/10 of the original volume.
EXAMPLE 2
Production of a D-xylulose/D-ribulose Mixture
Corynebacterium sp. ATCC 21050 was cultivated in a shake flask
under aerobic conditions at 30.degree. C. in a medium containing in
tap water 100 g/l glucose, 5 g/l yeast extract, 6 g/l urea, 30 ug/l
biotin, 10 g/l MgSO.sub.4 x7H.sub.2 O and 20 g/l KH.sub.2 PO.sub.4.
The pH was adjusted to 8 before inoculation with an overnight
aerobic culture in a medium containing 20 g/l glucose, 10 g/l yeast
extract, 10 g/l peptone and 2.5 g/l NaCl. After 120 hours the
glucose was converted to 13.6 g/l D-ribulose and 12.2 g/l
D-xylulose.
EXAMPLE 3
Enrichment of D-xylulose Through Epimerisation
Epimerisation was performed on the clarified concentrate obtained
in example 1. The pH of the concentrate was adjusted to 7.5, 10
units/ml of tagatose epimerase was added and the reaction was
carried out for 2 hours at 30.degree. C. The tagatose epimerase was
produced as described in Itoh et al. (1994). 71.2 g/l D-xylulose
and 46.6 g/l D-ribulose was analyzed in the reaction mixture after
the 2 hours.
EXAMPLE 4
Chromatographic Separation of Ribulose and Xylulose
An epimerisation reaction mixture containing ribulose and xylulose
was purified by applying chromatographic separation. The ribulose
content was about 14% on DS (dry substance) and the xylulose
content about 81% on DS. The rest or about 5% on DS comprised salts
and neutral compounds.
The separation was made in a laboratory scale column (diameter 0.1
m) as a batch process. A strongly acid cation exchange resin in
Ca.sup.2+ form was used, the bed height was about 1.5 m. The
cross-linkage degree of the resin was 5.5% and the average particle
size 0.3 mm. A feed having a concentration of 35 g/100 ml was used.
The separation temperature was 65.degree. C. and the flow rate 50
ml/min. The separation was performed as follows:
Step 1.
About 700 ml of feed solution was introduced to the top of the
resin bed. The feed and the column were preheated to 65.degree.
C.
Step 2.
The feed solution was eluted downwards the column by feeding
deionised water to the top of the resin bed. The eluent was also
preheated to 65.degree. C. The flow rate was controlled by an
outflow pump.
Step 3.
The outflow of the column was monitored continuously by on-line dry
substance (refractive index) device. The outflow was collected in
separate fractions at 2 min interval.
Step 4.
The composition of the collected samples was analyzed with HPLC.
According to this data, the outflow was pooled in two fractions and
a capacity calculation of these two product fractions was made.
Table 1 presents the composition of the feed solution and the
outflow fractions (purity and yield). Xylulose is eluting out
faster than ribulose, but the higher amount of xylulose resulted in
some overlapping of the profiles. In addition to this, the salts
are mainly eluting into the xylulose fraction reducing the purity
to some extent. Other neutral components are eluting in both
product fractions.
The yield is calculated by dividing the amount of the target
component in the target fraction by the amount of the target
component in both outcoming fractions.
TABLE 1 Composition of feed solution and outflow fractions Xylulose
Ribulose purity, % on purity, % on Xylulose Ribulose DS DS yield, %
yield, % Feed solution 14 81 -- -- Xylulose fraction 96 2 90 10
Ribulose fraction 34 53 10 90
The xylulose fraction was isomerised to produce xylose, and the
ribulose fraction was recirculated back to epimerisaton (for
xylulose production).
EXAMPLE 5
Chromatographic Separation of Ribulose and Xylulose
An epimerisation reaction mixture containing ribulose and xylulose
was purified by applying chromatographic separation. The ribulose
content was about 15% on DS and the xylulose content about 85% on
DS.
The separation was made in a laboratory scale column (diameter 0.1
m) as a batch process. A strongly acid cation exchange resin in
Ca.sup.2+ form was used, the bed height was about 1.5 m. The
cross-linkage degree of the resin was 5.5% and average particle
size 0.3 mm. A feed having a concentration of 35 g/100 ml was used.
The separation temperature was 65.degree. C. and the flow rate 50
ml/min. The separation was performed as follows:
Step 1.
About 700 ml of feed solution was introduced to the top of the
resin bed. The feed and the column were preheated to 65.degree.
C.
Step 2.
The feed solution was eluted downwards in the column by feeding
deionised water to the top of the resin bed. The eluent was also
preheated to 65.degree. C. The flow rate was controlled by an
outflow pump.
Step 3.
The outflow of the column was monitored continuously by on-line dry
substance (refractive index) device. The outflow was collected in
separate fractions at 2 min interval.
Step 4.
The composition of the pooled samples was analyzed with HPLC and a
capacity calculation of the two product fractions made.
Table 2 presents the composition of the feed solution and the
outflow fractions (purity and yield) calculated as described in
example 4. Elution behaviour was similar as in example 4.
TABLE 2 Composition of feed solution and outflow fractions Xylulose
Ribulose purity, % on purity, % Xylulose Ribulose DS on DS yield, %
yield, % Feed solution 15 85 -- -- Xylulose 98 2 90 10 fraction
Ribulose 39 61 10 90 fraction
EXAMPLE 6
Chromatographic Separation of Ribulose and Xylulose
An epimerisation reaction mixture containing ribulose and xylulose
was purified by applying chromatographic separation. The ribulose
content was about 38% on DS and the xylulose content about 58% on
DS. The rest or about 4% on DS comprised salts and other neutral
compounds.
The separation was made in a laboratory scale column (diameter 0.1
m) as a batch process. A strongly acid cation exchange resin in
Ca.sup.2+ form was used, the bed height was about 1.5 m. The
cross-linkage degree of the resin was 5.5% and the average particle
size 0.3 mm. A feed having a concentration of 35 g/100 ml was used.
The separation temperature was 65.degree. C. and the flow rate 50
ml/min. The separation was performed as follows:
Step 1.
About 700 ml of feed solution was introduced to the top of the
resin bed. The feed and the column were preheated to 65.degree.
C.
Step 2.
The feed solution was eluted downwards the column by feeding
deionised water to the top of the resin bed. The eluent was also
preheated to 65.degree. C. The flow rate was controlled by an
outflow pump.
Step 3.
The outflow of the column was monitored continuously by on-line dry
substance (refractive index) device. The outflow was collected in
separate fractions at 2 min interval.
Step 4.
The composition of the collected samples was analyzed with HPLC.
According to this data, the outflow was collected in two pools and
a capacity calculation of these two product fractions was made.
Table 3 presents the composition of the feed solution and the
outflow fractions (purity and yield). Xylulose and ribulose were
eluted similary as in example 4. Salts are mainly eluting into the
xylulose fraction and other neutral components are eluting into
both product fractions. Yields are calculated similarly as in
example 4.
TABLE 3 Composition of feed solution and outflow fractions Xylulose
Ribulose purity, % on purity, % on Xylulose Ribulose DS DS yield, %
yield, % Feed solution 38 58 -- -- Xylulose fraction 90 7 90 10
Ribulose fraction 14 81 10 90
EXAMPLE 7
Chromatographic Separation of Ribulose, Xylulose and Xylose
A reaction mixture containing ribulose, xylulose and xylose
(produced according to the methods disclosed in example 10 or 11)
was purified by applying chromatographic separation. The feed
solution had a xylose purity of about 67% on DS and a ribulose and
xylulose purity of about 14% on DS each. Some salts and neutral
components (about 5% on DS in total) were also found.
The separation was made in a laboratory scale column (diameter 0.1
m) as a batch process. A strongly acid cation exchange resin in
Ca.sup.2+ form was used, the bed height was about 1.5 m. The
cross-linkage degree of the resin was 5.5% and the average particle
size 0.3 mm. A feed having a concentration of 35 g/100 ml was used.
The separation temperature was 65.degree. C. and the flow rate 50
ml/min. The separation was performed as follows:
Step 1.
About 700 ml of feed solution was introduced to the top of the
resin bed. The feed and the column were preheated to 65.degree.
C.
Step 2.
The feed solution was eluted downwards the column by feeding
deionised water to the top of the resin bed. The eluent was also
heated to 65.degree. C. The flow rate was controlled by an outflow
pump.
Step 3.
The outflow of the column was monitored continuously by on-line dry
substance (refractive index) and conductivity measurement device.
The outflow was collected in separate fractions at 2 min
interval.
Step 4.
The composition of the collected samples was analyzed with HPLC.
According to this data, three fractions were pooled and a capacity
calculation of these product fractions made.
Table 4 presents the composition of the feed solution and the three
outflow fractions (purity and yield). Xylose is eluting out first,
but the high amount of xylose in the feed resulted in an
overlapping of xylose and xylulose. Salts are also eluting with the
xylose fraction. Ribulose is eluting as a last component and only a
small overlapping of xylulose and ribulose occurred. The other
neutral components were left under xylulose and ribulose peaks.
The yield is calculated by dividing the amount of the target
component in the target fraction by the amount of the target
component in all outcoming fractions.
TABLE 4 Composition of feed solution and outflow fractions Xylose
Xylulose Ribulose purity, purity, purity, Xylose yield, Xylulose
Ribulose % on DS % on DS % on DS % yield, % yield, % Feed solution
67 14 14 -- -- -- Xylose fraction 91 3 0 65 10 0 Xylulose 63 32 2
35 85 5 fraction Ribulose 0 5 89 0 5 95 fraction
The xylulose fraction was used in an isomerisation reaction to
produce xylose. The ribulose fraction was recirculated back to
epimerisation (for xylulose production). From the xylose fraction,
xylose was recovered by crystallization.
EXAMPLE 8
Chromatographic Separation of Ribulose, Xylulose and Xylose
A reaction mixture containing ribulose, xylulose and xylose was
purified by applying chromatographic separation. The feed solution
had a xylose purity of about 67% on DS, and a ribulose and xylulose
purity of about 14% on DS, each. Some salts and neutral components
(about 5% on DS in total) were also found.
The separation was made in a laboratory scale column (diameter 0.1
m) as a batch process. A strongly acid cation exchange resin in
Ca.sup.2+ form was used, the bed height was about 1.5 m. The
cross-linkage degree of the resin was 5.5% and the average particle
size 0.3 mm. A feed having a concentration of 35 g/100 ml was used.
The separation temperature was 65.degree. C. and the flow rate 50
ml/min. The separation was performed as follows:
Step 1.
About 700 ml of feed solution was introduced to the top of the
resin bed. The feed and the column were preheated to 65.degree.
C.
Step 2.
The feed solution was eluted downwards the column by feeding
deionised water to the top of the resin bed. The eluent was also
preheated to 65.degree. C. The flow rate was controlled by an
outflow pump.
Step 3.
The outflow of the column was monitored continuously by on-line dry
substance (refractive index) and conductivity measurement device.
The outflow was collected in separate fractions at 2 min
interval.
Step 4.
The composition of the collected samples was analyzed with HPLC.
According to this data, two fractions were pooled and a capacity
calculation of these product fractions made (xylose fraction and
xylulose+ribulose rich fraction).
Table 5 presents the composition of the feed solution and the two
outflow fractions (purity and yield). Again, xylose is eluting out
first, but the high amount of xylose in the feed resulted in some
overlapping of xylose and later eluting compounds. Salts are mainly
eluting in the xylose fraction. Ribulose is eluting as a last
component but it was collected in the same fraction with xylulose
and some other neutral components.
The yields are calculated as in example 7.
TABLE 5 Composition of feed solution and outflow fractions Xylose
Xylulose Ribulose purity, purity, purity, Xylose Xylulose Ribulose
% on DS % on DS % on DS yield, % yield, % yield, % Feed solution 67
14 14 -- -- -- Xylose fraction 91 3 0 65 10 0 Xylulose + 45 24 27
35 90 100 ribulose fraction
The xylose fraction was used in a hydrogenation reaction to produce
xylitol. The other product fraction was circulated back to
epimerisation to produce more xylulose.
EXAMPLE 9
Chromatographic Separation of Ribulose, Xylulose and Xylose
A reaction mixture containing ribulose, xylulose and xylose was
purified by applying chromatographic separation. The feed solution
had a xylose purity of about 70% on DS, and a ribulose and xylulose
purity of about 15% on DS each.
The separation was made in a laboratory scale column (diameter 0.1
m) as a batch process. A strongly acid cation exchange resin in
Ca.sup.2+ form was used, the bed height was about 1.5 m. The
cross-linkage degree of the resin was 5.5% and average particle
size 0.3 mm. A feed having a concentration of 35 g/100 ml was used.
Separation temperature was 65.degree. C. and flow rate 50 ml/min.
The separation was performed as follows:
Step 1.
About 700 ml of feed solution was introduced to the top of the
resin bed. The feed and the column were preheated to 65.degree.
C.
Step 2.
The feed solution was eluted downwards in the column by feeding
deionised water to the top of the resin bed. The eluent was also
preheated to 65.degree. C. The flow rate was controlled by an
outflow pump.
Step 3.
The outflow of the column was monitored continuously by on-line
substance (refractive index) device. The outflow was collected in
separate fractions at 2 min interval.
Step 4.
The composition of the collected samples was analyzed with HPLC and
capacity calculation of the three product fractions made.
Table 6 presents the composition of the feed solution and the
outflow flow fractions (purity and yield) calculated as in example
4. Xylose is eluting out first, but the high amount of xylose in
feed resulted in an overlapping of xylose and xylulose. Ribulose is
eluting as a last component and only a small overlapping of
xylulose and ribulose occurred.
TABLE 6 Composition of feed solution and outflow fractions Xylose
Xylulose Ribulose purity, purity, purity, Xylose Xylulose Ribulose
% on DS % on DS % on DS yield, % yield, % yield, % Feed solution 70
15 15 -- -- -- Xylose 97 3 0 65 10 0 fraction Xylulose 64 34 35 35
85 5 fraction Ribulose 0 5 95 0 5 95 fraction
The xylulose fraction was used in isomerisation to produce xylose
and the ribulose fraction was sent back to epimerisation (for
xylulose production). The xylose fraction was used in hydrogenation
to produce xylitol.
EXAMPLE 10
Isomerisation of the D-xylulose to D-xylose
A D-xylulose fraction obtained through chromatographic separation
was concentrated to a concentration of 400 g/l xylulose. The pH of
the concentrate was adjusted to 7.0 and 30 U/ml glucose isomerase
(Sweetzyme, Novo Nordisk AS) was added. The reaction was carried
out at 45.degree. C. for 4 hours. 311 g/l D-xylose and 73 g/l
D-xylulose was analyzed in the mixture after the reaction.
EXAMPLE 11
Isomerisation of Xylulose Without Prior Separation
The solution after xylulose enrichment such as in example 3 can
also be isomerised with xylose (glucose) isomerase without prior
chromatographic separation of the sugars. To effect this, a
solution containing 43.2 g/l D-ribulose and 72.1 g/l D-xylulose was
isomerised for 2 hours at 60.degree. C. after addition of 1.66 g/l
of glucose isomerase (Sweetzyme, Novo). After the isomerisation
21.9 g/l D-xylulose, 48.3 g/l D-xylose, 40.8 g/l D-ribulose and 1.1
g/l D-ribose were analyzed by HPLC.
EXAMPLE 12
Production of a Mixture of Ribitol and Arabinitol by Hydrogenation
of Ribulose
Ribulose solution from the fractions described in example 4, 5 or 6
was purified by an ion exchange method as described in example 17
and reduced to ribitol and arabinitol by hydrogenating ribulose
syrup at a temperature of 100.degree. C. and a pressure of 45 bar
in an agitated batch autoclave using Raney-nickel as catalyst. The
catalyst load was 10% wet catalyst of total solids of the syrup.
The pH of the feed syrup was adjusted to 6 before the reaction. The
dry substance of the feed was 50%. Hydrogenation time was three
hours. The conversion of ribulose was over 90% and it yielded a
50/50 percent mixture of ribitol and arabinitol.
EXAMPLE 13
Production of a Mixture of Xylitol and Arabinitol by Hydrogenation
of Xylulose
Xylulose solution from the fractions described in example 4, 5 or 6
was purified by an ion exchange method as described in example 17
and hydrogenated to xylitol and arabinitol by reducing xylulose
syrup at a temperature of 100.degree. C. at 45 bar pressure in an
agitated batch autoclave. The catalyst load was 10% wet catalyst of
total solids of the syrup. The catalyst was Raney-nickel. The pH of
the feed syrup was adjusted to 6 before the reaction. The dry
substance of the feed was 50%. Hydrogenation time was three hours.
The conversion of xylulose was more than 90% and the product was a
50/50 percent mixture of xylitol and arabinitol
EXAMPLE 14
Production of a Mixture of Ribitol, Xylitol and Arabinitol by
Hydrogenation of Ribulose
Ribulose solution from the fractions described in example 4, 5 or 6
was purified by an ion exchange method as described in example 17
and reduced to ribitol, xylitol and arabinitol by hydrogenating
ribulose syrup at a temperature of 120.degree. C. and a pressure of
70 bar in an agitated batch autoclave using Raney-nickel (Chemcat J
10 GS) as catalyst. The catalyst load was 80% wet catalyst of total
solids of the syrup. The pH of the feed syrup was adjusted to 6
before the reaction. The dry substance of the feed was 50%.
Hydrogenation time was twenty hours. The conversion of ribulose
yielded 55% ribitol, 10% xylitol, 30% arabinitol and 3% others.
EXAMPLE 15
Production of a Mixture of Xylitol, Ribitol and Arabinitol by
Hydrogenation of Xylulose
Xylulose solution from the fractions described in example 4, 5 or 6
was purified by an ion exchange method as described in example 17
and hydrogenated to xylitol, ribitol and arabinitol by reducing
xylulose syrup at a temperature of 120.degree. C. and a pressure of
70 bar in an agitated batch autoclave. The catalyst load was 80%
wet catalyst of total solids of the syrup. The catalyst was
Raney-nickel. The pH of the feed syrup was adjusted to 6 before the
reaction. The dry substance of the feed was 50%. Hydrogenation time
was 24 hours. The conversion of xylulose yielded 60% xylitol, 30%
arabinitol, 8% ribitol and 2% others.
EXAMPLE 16
Chromatographic Separation of Xylose and Xylulose
An isomerisation reaction mixture containing xylose and xylulose
(obtained according to the method described in examples 4 and 10)
was purified by applying chromatographic separation. The xylose
content was about 78% on DS and the xylulose content about 17% on
DS. The rest or about 5% on DS comprised salts and neutral
compounds.
The separation was made in a laboratory scale column (diameter 0.1
m) as a batch process. A strongly acid cation exchange resin in
Ca.sup.2+ form was used, the bed height was about 1.5 m. The
cross-linkage degree of the resin was 5.5% and the average particle
size 0.3 mm. A feed having a concentration of 35 g/100 ml was used.
The separation temperature was 65.degree. C. and the flow rate 50
ml/min. The separation was performed as follows:
Step 1.
About 700 ml of feed solution was introduced to the top of the
resin bed. The feed and the column were preheated to 65.degree.
C.
Step 2.
The feed solution was eluted downwards the column by feeding
deionised water to the top of the resin bed. The eluent was also
preheated to 65.degree. C. The flow rate was controlled by an
outflow pump.
Step 3.
The outflow of the column was monitored continuously by on-line dry
substance (refractive index) device. The outflow was collected in
separate fractions at 2 min interval.
Step 4.
The composition of the collected samples was analyzed with HPLC.
According to this data, the outflow was pooled in two fractions and
a capacity calculation of these two product fractions was made.
Table 7 presents the composition of the feed solution and the
outflow fractions (purity and yield). Xylose is eluting out faster
than xylulose, but the high amount of xylose resulted in some
overlapping of the profiles. In addition to this, the salts are
also eluting into the xylose fraction reducing the purity to some
extent. Other neutral components are eluting in both product
fractions.
Yields are calculated as in previous examples.
TABLE 7 Composition of feed solution and outflow fractions Xylose
Xylulose purity, % on purity, % Xylose Xylulose DS on DS yield, %
yield, % Feed solution 78 17 -- -- Xylose fraction 93 3 75 10
Xylulose fraction 53 42 25 90
The xylulose fraction was recirculated back to isomerisation to
produce more xylose, and the xylose fraction was used in a
hydrogenation reaction to produce xylitol.
EXAMPLE 17
Purification of Xylose
The xylose product of a ribulose/xylulose/xylose-separation process
according to example 9 was purified before the conversion step. The
purification was made using a strongly acid cation exchange resin
(Purolite C 150) and a weakly basic anion exchange resin (Dow 66).
The temperature during the purification step was 40.degree. C. and
the flow rate 2-3 bed volumes in hour. The syrup concentration was
150 9/1.
EXAMPLE 18
Hydrogenation of Xylose to Xylitol
The purified xylose obtained in example 14 was subjected to a
hydrogenation reaction. The hydrogenation was carried out in a
stirred batch autoclave. The hydrogen pressure was 40 bar and the
temperature 110.degree. C. The mixing speed was 800 rpm. As
catalyst, Raney nickel was used in a dosage of 10% wet catalyst per
syrup dry substance. The hydrogenation time was three hours, the
reducing sugar content after hydrogenation was <0.1% and the
xylitol content 95.5%. The xylitol produced can be recovered e.g.
as a crystalline product (as described by Jaffe in U.S. Pat. No.
4,066,711).
* * * * *